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EP-3748005-B1 - TRANSGENIC METHOD OF OBTAINING BLUE FLOWERS BY CATALYZING GLUTAMINE TO SYNTHESIZE INDIGO

EP3748005B1EP 3748005 B1EP3748005 B1EP 3748005B1EP-3748005-B1

Inventors

  • ZHANG, YAN
  • CHEN, YIHUA
  • ANKANAHALLI, Nanjaraj Urs
  • HU, Yiling
  • LI, Pengwei

Dates

Publication Date
20260506
Application Date
20181123

Claims (10)

  1. A transgenic method of obtaining a plant with a blue flower by catalyzing synthesis from glutamine to indigoidine comprising: transfecting a Sfp gene encoding phosphopantetheinyl transferase and a bpsA gene encoding indigoidine synthetase into the plant to allow Sfp and BpsA proteins to be functionally expressed in a petal cell of the plant, thereby turning a petal blue via synthesis from glutamine to indigoidine; and the Sfp gene is a gene that encodes phosphopantetheinyl transferase, or a gene that encodes an isoenzyme of phosphopantetheinyl transferase; the bpsA gene is a gene that encodes indigoidine synthetase, or a gene that encodes an isoenzyme of indigoidine synthetase; and the plant is a rose.
  2. The method of claim 1, characterized in that the Sfp gene has the nucleotide sequence as shown in SEQ ID NO. 1; and/or phosphopantetheinyl transferase encoded by the Sfp gene has the amino acid sequence as shown in SEQ ID NO. 2.
  3. The method of claim 1, wherein the bpsA gene has the nucleotide sequence as shown in SEQ ID NO. 3; and/or the indigoidine synthetase encoded by the bpsA gene has the amino acid sequence as shown in SEQ ID NO. 4.
  4. The method of any one of claims 1 to 3, characterized in that the Sfp gene encoding the phosphopantetheinyl transferase and the bpsA gene encoding indigoidine synthetase are respectively cloned into a plasmid; and the plasmid contains plant promoters, and the Sfp gene and the bpsA gene are operably linked to one of the plant promoters, respectively.
  5. The method of claim 4, characterized in that the plant promoter upstream the Sfp gene is CHSp, which has the nucleotide sequence as shown in SEQ ID NO. 5; and/or the plant promoter upstream the bpsA gene is RhAGp, which has the nucleotide sequence as shown in SEQ ID NO. 6.
  6. The method of any one of claims 4 to 5, characterized in that the plasmid is amplified in Escherichia coli and the amplified plasmid is transformed into Agrobacterium tumefaciens.
  7. The method of any one of claims 1 to 6, characterized in that the Sfp gene and the bpsA gene are transfected into the plant based on Agrobacterium- mediated transfection.
  8. The method of any one of claims 1 to 7, characterized in that the plant is Rosa chinensis.
  9. A plant, which is transfected with a Sfp gene encoding phosphopantetheinyl transferase and a bpsA gene encoding indigoidine synthetase, such that a phosphopantetheinyl transferase encoded by the Sfp gene and indigoidine synthetase encoded by the bpsA gene are functionally expressed in a petal cell of the plant, thereby turning a petal blue via synthesis from glutamine to indigoidine; and the Sfp gene is a gene that encodes phosphopantetheinyl transferase, or a gene that encodes an isoenzyme of phosphopantetheinyl transferase; the bpsA gene is a gene that encodes indigoidine synthetase, or a gene that encodes an isoenzyme of indigoidine synthetase; and the plant is a rose.
  10. The plant of claim 9, characterized in that the plant is Rosa chinensis.

Description

TECHNICAL FIELD The invention belongs to the field of biotechnology, and relates to a transgenic method of obtaining a plant with blue flowers by catalyzing synthesis from glutamine to indigoidine. BACKGROUND TO THE INVENTION Flower color is an important ornamental feature of ornamental plants, and improvement in flower color has always been one of the important goals of gardeners. The color of flowers not only plays an important role in the pollination and reproduction of plants, but also provides a colorful viewing experience for humans, and thus has important aesthetic value (Grotewold, E. The genetics and biochemistry of floral Pigments [J]. Annual Review of Plant Biology, 2006, 57(1): 761). As an important ornamental plant, modern rose (Rosa hybrida) has been cultivated for 5000 years. So far, more than 2,500 varieties have cultivated, but there are no true blue roses among them. Actually, the existing blue roses on the market, such as "Blue Fairy", are dyed blue with dyes (Holton T.A, Tanaka Y. Blue roses: a pigment of our imagination? [J]. Trends in Biotechnology: United Kingdom, 1994, 12(2): 40-42). Blue rose as an idiom in English means a miracle that would never come true. It frequently appears in various literary and artistic works including movies, TV, poetry, novels, dramas and the like. The flower color of a plant is determined by many factors, including flavonoids such as anthocyanins, terpenes such as carotene, as well as pH of vacuole and metal ions, etc. (Tanaka Y, Brugliera F. Flower colour and cytochromes P450 [J]. Philosophical Transactions of the Royal Society of London, 2013, 368 (1612): 283-291). The hydroxylation of anthocyanins at different positions and different degrees makes the flowers have different colors, in which the flavonoid 3'5' hydroxylase (F3'5'H) catalyzes the formation of delphinidin, which leads to blue flowers for many plants. Since many plants including roses lack the gene encoding F3'5'H, such plants do not have blue flowers (Mikanagi Y, Saito N, Yokoi M, et al. Anthocyanins in flowers of genus Rosa, sections Cinnamomeae (=Rosa), Chinenses, Gallicanae and some modern garden roses [J]. Biochemical Systematics & Ecology, 2000, 28(9): 887). Suntory Corp. from Japan used transgenic technology to make plants that cannot bloom blue flowers in nature bloom blue flowers by introducing foreign genes including F3'5'H and simultaneously suppressing the expression of several other genes. This method involves the modifications to multiple genes, and requires complex precursor materials, and therefore the cost is expensive. This technology has been successful in carnations and chrysanthemums. Since the pH of vacuole in roses is very low (about 2.7), this technology can only obtain lilac flowers for roses, and cannot show the true blue color (Figure 1) (Katsumoto Y, Fukuchimizutani M, Fukui Y, et al. Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. [J]. Plant & Cell Physiology, 2007, 48(11): 1589). Nonetheless, each of the lilac roses costs 22-35 US dollars (Staff (20 October 2009). "Blue roses to debut in Japan". The Independent, House and Home.Retrieved 30 August 2012), and it brings huge economic benefits to Suntory. Currently, the roses are only available in Japan and the American continent. The acquisition of true blue roses is still a technical problem that has not been solved yet. SUMMARY OF THE INVENTION The purpose of the present invention is to overcome the deficiencies of the prior art and provides a transgenic method of obtaining a plant with blue flowers by catalyzing synthesis from glutamine to indigoidine. The technical solution of the present invention is summarized as follows: A transgenic method of obtaining a plant with a blue flower by catalyzing synthesis from glutamine to indigoidine includes the following steps: 1) selecting a Sfp gene encoding phosphopantetheinyl transferase and a bpsA gene encoding indigoidine synthetase, and cloning the two genes downstream a plant promoter of a plasmid containing the plant promoter, respectively;2) amplifying the plasmid obtained in step 1) in Escherichia coli and transforming the amplified plasmid into Agrobacterium tumefaciens;3) transfecting Sfp and bpsA DNA into the plant by Agrobacterium-mediated transfection;4) allowing Sfp and BpsA proteins to be functionally expressed in a petal cell, thereby turning a petal blue via synthesis from glutamine to indigoidine; wherein the Sfp gene is a gene derived from any species that encodes phosphopantetheinyl transferase, or a gene that encodes an isoenzyme of phosphopantetheinyl transferase; andthe bpsA gene is a gene derived from any species that encodes indigoidine synthetase, or a gene that encodes an isoenzyme of indigoidine synthetase; The plant promoter is a DNA sequence that can drive downstream gene expression in plant cells. Preferably, the Sfp gene has the nucleotide sequence as shown in SEQ